CN107177672B - Standard gene for identifying disc fungi and application thereof - Google Patents
Standard gene for identifying disc fungi and application thereof Download PDFInfo
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- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 36
- 241000233866 Fungi Species 0.000 title abstract description 10
- 238000000034 method Methods 0.000 claims abstract description 19
- 241000894007 species Species 0.000 claims abstract description 18
- 241001264635 Discodermia Species 0.000 claims abstract description 12
- 238000003752 polymerase chain reaction Methods 0.000 claims abstract description 10
- 230000003321 amplification Effects 0.000 claims abstract description 8
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 8
- 241000894006 Bacteria Species 0.000 claims description 11
- 238000000137 annealing Methods 0.000 claims description 9
- 238000012163 sequencing technique Methods 0.000 claims description 8
- 238000004925 denaturation Methods 0.000 claims description 4
- 230000036425 denaturation Effects 0.000 claims description 4
- 230000008014 freezing Effects 0.000 claims description 4
- 238000007710 freezing Methods 0.000 claims description 4
- 239000002773 nucleotide Substances 0.000 claims description 4
- 125000003729 nucleotide group Chemical group 0.000 claims description 4
- 238000012257 pre-denaturation Methods 0.000 claims description 4
- 230000000877 morphologic effect Effects 0.000 abstract description 6
- 241000244206 Nematoda Species 0.000 abstract description 5
- 239000011543 agarose gel Substances 0.000 abstract description 2
- 241001465754 Metazoa Species 0.000 abstract 1
- 238000001514 detection method Methods 0.000 abstract 1
- 210000003470 mitochondria Anatomy 0.000 abstract 1
- 230000002438 mitochondrial effect Effects 0.000 abstract 1
- 108020004414 DNA Proteins 0.000 description 12
- 239000012634 fragment Substances 0.000 description 6
- 230000002068 genetic effect Effects 0.000 description 3
- 238000007621 cluster analysis Methods 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 241000722808 Arthrobotrys Species 0.000 description 1
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical class [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 1
- 102000012286 Chitinases Human genes 0.000 description 1
- 108010022172 Chitinases Proteins 0.000 description 1
- 241001430149 Clostridiaceae Species 0.000 description 1
- 102000029816 Collagenase Human genes 0.000 description 1
- 108060005980 Collagenase Proteins 0.000 description 1
- 241001272122 Dactylellina Species 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108020005196 Mitochondrial DNA Proteins 0.000 description 1
- 241000214023 Orbiliaceae Species 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 102000012479 Serine Proteases Human genes 0.000 description 1
- 108010022999 Serine Proteases Proteins 0.000 description 1
- 108010006785 Taq Polymerase Proteins 0.000 description 1
- 230000003044 adaptive effect Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 229960002424 collagenase Drugs 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- ZQTNQVWKHCQYLQ-UHFFFAOYSA-N dronedarone Chemical compound C1=CC(OCCCN(CCCC)CCCC)=CC=C1C(=O)C1=C(CCCC)OC2=CC=C(NS(C)(=O)=O)C=C12 ZQTNQVWKHCQYLQ-UHFFFAOYSA-N 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 244000000004 fungal plant pathogen Species 0.000 description 1
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L magnesium chloride Substances [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
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- 230000007923 virulence factor Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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Abstract
The invention relates to a standard gene sequence for a discodermia and an identification method, belonging to the field of species molecular identification. The standard detection mitochondria mtLSU gene has a gene sequence SEQ ID NO. 1. Polymerase Chain Reaction (PCR) amplification was performed using the mitochondrial large subunit mtLSU gene universal primers ML7, ML8, followed by specific primer sequences ML-chaoF: AGAACAACGGCTAGGTGG, ML-chaoR: GAACGAGTCCGCAGAAGA, and the PCR product is verified by agarose gel and sequenced. According to the sequence difference of mtLSU gene, the discodermia can be quickly and accurately identified. The invention adopts a reliable DNA molecular identification technology, does not need to consume a large amount of time to carry out morphological identification on the entomogenous fungi, can quickly and accurately identify the entomogenous fungi and provides a powerful research tool for preventing and controlling animal and plant nematodes.
Description
Technical Field
The invention relates to the field of species molecular identification, in particular to a standard gene for identifying a discodermia and application thereof
Background
(of the family Clostridiaceae) ((Orbiliaceae) Is a small-sized discodermus widely distributed in the world and is a nutrient saprophytic life. In a long history of evolution, the mycelium is adapted to the living environment, can be transformed into various kinds of predatory organ predatory nematodes with delicate structures, and can also be transformed to parasitize other plant pathogenic fungi. In addition, some virulence factors, such as extracellular enzymes including serine proteases, chitinase and collagenase, can be produced and are involved directly or indirectly in the process of infecting the nematode body walls. The fungi are important factors for natural control of nematode population, are also important research materials for biological control of plant diseases, and have great research on biological control and adaptive evolutionAnd (4) potential. The family of fungi now contains several important genera of nematophagous hyphomycete anamorphs, e.g.Arthrobotrys、Dactylellina、DrechslerellaAnd the like. However, the fungus of the family contains various types of anamorph, has various species morphological characteristics, has no obvious microstructure difference among some species, is insufficient in molecular phylogenetic research of the fungus at present, and causes great obstacles to classification, scientific nomenclature and diversity research due to the lack of molecular evidence and species definition standards. Therefore, there is an urgent need for standard genes to establish rapid molecular identification, and the identification and classification problems are solved by combining morphological studies and molecular data.
Disclosure of Invention
In order to overcome the defects of morphological identification of the disc bacteria, the invention provides a standard gene sequence for molecular identification of the disc bacteria and an identification method, which can quickly and accurately identify the confusable species of the disc bacteria, thereby providing a quick and effective identification tool for the prevention and treatment of nematodes.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a standard gene for identifying a disc bacterium molecule and application thereof comprise the steps of establishing an mtLSU standard gene fragment and identifying a sample; wherein:
(1) according to the technical principle of DNA bar codes, a PCR amplification method is adopted to ensure the purity and reliability of the strip. The sequencing result is manually corrected and sequence spliced, and the obtained sequence is ensured to be a standard sequence. Extracting genome DNA from the collected disc bacterium sample, performing Polymerase Chain Reaction (PCR) amplification by using primers to obtain mtLSU gene of the sample, sequencing the amplification product, analyzing and comparing, and taking the mtLSU gene sequence fragment of the sample amplified by the primer sequence of the SEQ ID NO.1 sample as a standard gene.
(2) Identification of the sample: comparing a sample sequence to be identified with a standard gene, establishing an identification rule based on homology and a phylogenetic tree method based on cluster analysis to identify species, and obtaining the similarity between the sequences, wherein the corresponding species with the highest similarity or the highest matching degree or the closest genetic distance is the species to be identified; the method comprises the following specific steps:
a. identification of species based on homology identification rules
According to the sequencing result, if the number of nucleotide differences with the gene sequence SEQ ID NO.1 is less than 10, the sample to be detected can be judged to be a certain species of the discodermus.
b. Clustering analysis-based phylogenetic tree method for establishing identification rule to identify species
Combining the standard gene sequence and the sample sequence to be identified with the mtLSU gene sequence of the discodermia, constructing an NJ phylogenetic tree by applying MEGA or PAUP software, detecting the species of which the DNA barcode sequence of the unidentified sample and the standard gene sequence are clustered together, and identifying the species if the unknown sample and the species in the database form a single-branch system.
The invention has the advantages that:
1. the mtLSU gene is preferably used as a DNA bar code most suitable for identifying the discodermia, compared with other genes, the mtLSU is a mitochondrial gene, and the sequence of the mtLSU has the characteristics of universality and easy comparison.
2. Establishes the standard gene sequence of the discodermia and the identification method of the sample, and greatly improves the identification efficiency compared with the traditional morphological identification method. The method has low requirement on the integrity of the sample, and the identification index can be quantified, thereby providing an effective basis for identifying the disc bacteria in time. In addition, the reliability and accuracy of the identification are greatly superior to those of the conventional molecular identification method by setting an identification rule for identification by using a phylogenetic tree method based on cluster analysis, so that the defect of the identification of the disc bacteria molecules is overcome.
Description of the drawings:
the attached figure is an NJ tree constructed based on the Kimura-2-parameter model genetic distance of a sample sequence to be identified and mtLSU standard gene sequences of all the genera of the discodermia.
The specific implementation mode is as follows:
the invention is further illustrated below with reference to specific examples:
example 1: homology identification of sample to be detected and disc bacterium DNA bar code identification standard gene fragment
1. Collecting and preserving a disc fungus specimen: 10 samples from Yunnan province were collected.
2. Extracting the genome DNA of the disc fungus: genomic DNA was extracted using a modified CTAB method and the DNA concentration of the sample was diluted to 0.5. mu.g/. mu.l with sterilized deionized water.
3. Amplifying DNA fragments and carrying out Polymerase Chain Reaction (PCR), namely, amplifying by using universal primers, wherein the sequence of an outer primer pair is as follows:
forward primer ML7: 5 'GACCCTATGCAGCTTCTACTG 3';
reverse primer ML8: 5 'TTATCCCTAGCGTAACTTTTATC 3';
4. the specific primer according to claim 2, wherein the inner primer sequence is:
a forward primer ML-chaoF, 5 'AGAACAACGGCTAGGTGG 3';
the reverse primer ML-chaoR is 5 'GAACGAGTCCGCAGAAGA 3'.
The PCR reaction system was 25. mu.l: ddH2O18μl、PCR Buffer 2.5μl、MgCl22.5 mul, 0.5 mul dNTPs, 0.5 mul Taq DNA polymerase and 1 mul DNA template without dye; outer primer amplification procedure: pre-denaturation at 95 deg.C for 5min, denaturation at 94 deg.C for 40s, annealing at 37 deg.C for 55s, annealing at low temperature for 2-3 cycles, freezing the final annealing temperature at 50 deg.C, annealing at 55s and 72 deg.C for 1min, performing 30 cycles, extending at 72 deg.C for 10min, and storing at 4 deg.C. The amplification conditions of the inner primers are as follows: pre-denaturation at 95 deg.C for 5min, denaturation at 94 deg.C for 40s, annealing at 55-50.5 deg.C gradually reduced by 0.5 deg.C for 11 cycles, and final freezing at 50 deg.C, 25 cycles, and extension at 72 deg.C for 1min, and performing 30 cycles, final extension at 72 deg.C for 10min, and storing at 4 deg.C.
5. The amplified product was loaded and detected by electrophoresis in 1.0% agarose gel, 1 × TBE electrophoresis solution, and the size of PCR fragment was detected using DNAmarker. If a clear band with the size of 300-400bp appears and no hybrid band exists, the sequence is sent for sequencing.
6. Firstly, checking the quality of a sequence peak image obtained after sequencing in software Chromas, and splicing forward and reverse sequences by using SeqMan in a DNASTAR software package after determining that the quality of the peak image meets the requirement of data analysis. And (3) manually proofreading the sequencing result, splicing the sequence, and judging that the tissue to be detected is the discodermus if the difference number of the target gene fragment and the nucleotide of the gene sequence SEQ ID NO.1 is less than 10.
Example 2: identification rule established by phylogenetic tree method to identify species
1. Obtaining a sequence of a sample to be detected by adopting the steps described in the embodiment 1, combining a standard gene sequence, the sequence of the sample to be identified and an mtLSU sequence of the sample to be detected, applying MEGA (Mega-based genetic Algorithm) to repeat 1000 times based on a Kimura-2-parameter model to construct a phylogenetic tree, clustering DNA barcode sequences of unidentified samples and the standard gene sequence together, presenting obvious divergence in the sequences of 6 genera in the family of the pucheriaceae, and obtaining sequences 1 and 1 in a sequence tableArthrobotry musiformisWhen the samples are gathered together, the samples to be identified can be identified as the discodermia.
Compared with the traditional morphological identification method, the gene sequence obtained by the invention and the application method are beneficial to realizing the rapid and accurate identification of the disc bacteria.
SEQUENCE LISTING
<110> university of Yunnan
<120> standard gene for identifying disc bacteria and application thereof
<130> 2017
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 314
<212> DNA
<213> YMF1.03477 EQ ID NO.1
<400> 1
aagagtagct gggtatatcc taattccgaa aaatttgggg aaaaaattat attttttttc 60
ttttgagtct ttatttaata tataaaattt atataaattt aatatttcgc cgatcgccta 120
tggcggttga ccgatttctt aagatgaatt tatatataag aggataagat atattactta 180
tccctgtata tacttaatca ctatcttttt atttaaagta gaatttaatt ttaaagcaag 240
taagatttta actatatcga ggataggtgt aataaataaa attcacattt tctagtcttc 300
tgcggactcg ttca 314
Claims (3)
1. A DNA barcode standard gene of a discodermus is characterized in that the nucleotide sequence of the DNA barcode standard gene is a DNA molecular sequence shown as a sequence 1 in a sequence table.
2. The method for identifying the molecule of the discodermia paniculata by using the DNA barcode standard gene of the discodermia paniculata as described in claim 1, which comprises the following 4 steps:
(1) extracting genome DNA from sample hyphae;
(2) taking the genome DNA extracted in the step (1) as a template, amplifying by using a disc bacterium universal outer primer, and then carrying out polymerase chain reaction by using a specific inner primer, wherein the sequence of the outer primer is as follows: the forward primer ML7: 5 'GACCCTATGCAGCTTCTACTG 3', the reverse primer ML8: 5 'TTATCCCTAGCGTAACTTTTATC 3', the inner primer sequence is: a forward primer ML-chaoF of 5 'AGAACAACGGCTAGGTGG 3', a reverse primer ML-chaoR of 5 'GAACGAGTCCGCAGAAGA 3';
(3) sequencing the DNA product obtained by amplification in the step (2);
(4) and (3) manually splicing and correcting the sequencing result, carrying out homology comparison with a representative strain sequence SEQ ID NO.1 of the discodermia in the gene sequence table, and if the number of nucleotide differences is less than 10, identifying the sample as the discodermia.
3. The method for molecular identification of a pucheria species by using a DNA barcode standard gene of a pucheria species according to claim 2, wherein the amplification conditions of the outer primers are as follows: pre-denaturation at 95 ℃ for 5min, denaturation at 94 ℃ for 40s, annealing at 37 ℃ for 55s, annealing at low temperature for 2-3 cycles, freezing the final annealing temperature at 50 ℃, 55s, and extension at 72 ℃ for 1min, performing 30 cycles, extension at 72 ℃ for 10min, and storing at 4 ℃; the amplification conditions of the inner primers are as follows: pre-denaturation at 95 deg.C for 5min, denaturation at 94 deg.C for 40s, annealing at 55-50.5 deg.C gradually reduced by 0.5 deg.C for 11 cycles, and final freezing at 50 deg.C, 25 cycles, and extension at 72 deg.C for 1min, and performing 30 cycles, final extension at 72 deg.C for 10min, and storing at 4 deg.C.
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Patent Citations (6)
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